In 1967 I found that tRNA isolated from Tetrahymena mitochondria hybridized poorly with mitochondrial DNA. From competition- hybridization experiments between mitochondrial and cytoplasmic tRNAs, it was concluded that most mitochondrial tRNAs are nuclear transcripts and hence transported from the cytoplasm. Recent investigations in our laboratory reveal that, while three isoaccepting leucyl tRNA and two phenylalanyl tRNA species in mitochondria can form stable hybrids with mitochondrial DNA, arginyl, valyl, and lysyl tRNA species present in mitochondria do not hybridize with mDNA. In view of the profound importance of these results, it is desirable to extend and further substantiate them. Mitochondrial tRNAs two organisms, Tetrahymena and rat, will be thoroughly analyzed and compared for the extent of isoaccepting tRNA forms specifying the same amino acids by reversed-phase chromatography. These inoacceptors will then be individually tested to determine whether they are mitochondrial or nuclear DNA transcripts by hybrization tests. Furthermore, functional roles of these tRNAs will be examined by the specific codon bindin assay. These experiments should not only clarify the transcriptional nature and extent of the multiplicity of isoaccepting tRNA species, but also provide foundations for many biological phenomena includng development, differentiation, and neoplasia formation in which an involvement of tRNA has been implicated previously.